Adaptive laboratory evolution and genetic engineering improved terephthalate utilization in Pseudomonas putida KT2440.

Poly(ethylene terephthalate) (PET) is one of the most ubiquitous plastics and can be depolymerized through biological and chemo-catalytic routes to its constituent monomers, terephthalic acid (TPA) and ethylene glycol (EG). TPA and EG can be re-synthesized into PET for closed-loop recycling or microbially converted into higher-value products for open-loop recycling. Here, we expand on our previous efforts engineering and applying Pseudomonas putida KT2440 for PET conversion by employing adaptive laboratory evolution (ALE) to improve TPA catabolism.

Engineering of insect juvenile hormone III biosynthesis in the plant Nicotiana benthamiana.

Juvenile hormones (JHs) are farnesoic acid-derived sesquiterpenoids that play a crucial role in regulating various developmental processes in insects. Based on these reported biological activities, JHs and their synthetic analogs have been utilized as insecticides with significant commercial success over the past years. Here we describe the engineering of the JH pathway of the yellow fever mosquito (Aedes aegypti) by transient gene expression in the plant Nicotiana benthamiana.

De novo 2'-fucosyllactose biosynthesis using glucose as the sole carbon source by multiple engineered Bacillus subtilis.

2'-Fucosyllactose (2'-FL) is the most abundant human milk oligosaccharide and plays significant roles in gut microbiome balance, neural development, and immunoregulation. However, current fermentation schemes using multiple carbon sources increase production cost and metabolism burden. This study reported the development of an engineered Bacillus subtilis strain that produces 2'-FL using glucose as the sole carbon source.

Construction of a synthetic metabolic pathway for biosynthesis of threonine from ethylene glycol.

Ethylene glycol is a promising substrate for bioprocesses which can be derived from widely abundant CO or plastic waste. In this work, we describe the construction of an eight-step synthetic metabolic pathway enabling carbon-conserving biosynthesis of threonine from ethylene glycol. This route extends the previously disclosed synthetic threose-dependent glycolaldehyde assimilation (STEGA) pathway for the synthesis of 2-oxo-4-hydroxybutyrate with three additional reaction steps catalyzed by homoserine transaminase, homoserine kinase, and threonine synthase.

Deciphering molecular drivers of lactate metabolic shift in mammalian cell cultures.

Lactate metabolism plays a critical role in mammalian cell bioprocessing, influencing cellular performance and productivity. The transition from lactate production to consumption, known as lactate metabolic shift, is highly beneficial and has been shown to extend culture lifespan and enhance productivity, yet its molecular drivers remain poorly understood. Here, we have explored the mechanisms that underpin this metabolic shift through two case studies, illustrating environmental- and genetic-driven factors.

Model-assisted CRISPRi/a library screening reveals central carbon metabolic targets for enhanced recombinant protein production in yeast.

Production of recombinant proteins is regarded as an important breakthrough in the field of biomedicine and industrial biotechnology. Due to the complexity of the protein secretory pathway and its tight interaction with cellular metabolism, the application of traditional metabolic engineering tools to improve recombinant protein production faces major challenges. A systematic approach is required to generate novel design principles for superior protein secretion cell factories.

Elucidation and engineering of Sphingolipid biosynthesis pathway in Yarrowia lipolytica for enhanced production of human-type sphingoid bases and glucosylceramides.

Sphingolipids are vital membrane components in in mammalian cells, plants, and various microbes. We aimed to explore and exploit the sphingolipid biosynthesis pathways in an oleaginous and dimorphic yeast Yarrowia lipolytica by constructing and characterizing mutant strains with specific gene deletions and integrating exogenous genes to enhance the production of long-chain bases (LCBs) and glucosylceramides (GlcCers). To block the fungal/plant-specific phytosphingosine (PHS) pathway, we deleted the SUR2 gene encoding a sphinganine C4-hydroxylase, resulting in a remarkably elevated secretory production of dihydrosphingosine (DHS) and sphingosine (So) without acetylation.

Metabolic engineering of Escherichia coli for N-methylserotonin biosynthesis.

N-methylserotonin (NMS) is a valuable indole alkaloid with therapeutic potential for psychiatric and neurological disorders, and it is used in health foods, cosmetics, and weight loss supplements. However, environmental challenges and low reaction efficiencies significantly hinder cost-effective, large-scale production of NMS in plants or through chemical synthesis. Herein, we have successfully engineered Escherichia coli strains to enhance NMS production from L-tryptophan using whole-cell catalysis.

Optimized production of concanamycins using a rational metabolic engineering strategy.

Plecomacrolides, such as concanamycins and bafilomycins, are potent and specific inhibitors of vacuolar-type ATPase. Concanamycins are 18-membered macrolides with promising therapeutic potential against multiple diseases, including viral infection, osteoporosis, and cancer. Due to the complexity of their total synthesis, the production of concanamycins is only achieved through microbial fermentation.

Network for knowledge Organization (NEKO): An AI knowledge mining workflow for synthetic biology research.

Large language models (LLMs) can complete general scientific question-and-answer, yet they are constrained by their pretraining cut-off dates and lack the ability to provide specific, cited scientific knowledge. Here, we introduce Network for Knowledge Organization (NEKO), a workflow that uses LLM Qwen to extract knowledge through scientific literature text mining. When user inputs a keyword of interest, NEKO can generate knowledge graphs to link bioinformation entities and produce comprehensive summaries from PubMed search.

Flux balance analysis and peptide mapping elucidate the impact of bioreactor pH on Chinese hamster ovary (CHO) cell metabolism and N-linked glycosylation in the fab and Fc regions of the produced IgG.

Culture conditions have a profound impact on therapeutic protein production and glycosylation, a critical therapeutic-quality attribute, especially for monoclonal antibodies (mAbs). While the critical culture parameter of pH has been known since the early 1990s to affect protein glycosylation and production, detailed glycan and metabolic characterization and mechanistic understanding are critically lacking. Here, Chinese Hamster Ovary (CHO) cells were grown in bioreactors at pH 6.

Deep learning for NAD/NADP cofactor prediction and engineering using transformer attention analysis in enzymes.

Understanding and manipulating the cofactor preferences of NAD(P)-dependent oxidoreductases, the most widely distributed enzyme group in nature, is increasingly crucial in bioengineering. However, large-scale identification of the cofactor preferences and the design of mutants to switch cofactor specificity remain as complex tasks. Here, we introduce DISCODE (Deep learning-based Iterative pipeline to analyze Specificity of COfactors and to Design Enzyme), a novel transformer-based deep learning model to predict NAD(P) cofactor preferences.

Unraveling productivity-enhancing genes in Chinese hamster ovary cells via CRISPR activation screening using recombinase-mediated cassette exchange system.

Chinese hamster ovary (CHO) cells, which are widely used for therapeutic protein production, have been genetically manipulated to enhance productivity. Nearly half of the genes in CHO cells are silenced, which are promising targets for CHO cell engineering. To identify novel gene targets among the silenced genes that can enhance productivity, we established a genome-wide clustered regularly interspaced short palindromic repeats activation (CRISPRa) screening platform for bispecific antibody (bsAb)-producing CHO (CHO-bsAb) cells with 110,979 guide RNAs (gRNAs) targeting 13,812 silenced genes using a virus-free recombinase-mediated cassette exchange-based gRNA integration method.

Versatile xylose and arabinose genetic switches development for yeasts.

Inducible transcription systems are essential tools in genetic engineering, where tight control, strong inducibility and fast response with cost-effective inducers are highly desired. However, existing systems in yeasts are rarely used in large-scale fermentations due to either cost-prohibitive inducers or incompatible performance. Here, we developed powerful xylose and arabinose induction systems in Saccharomyces cerevisiae, utilizing eukaryotic activators XlnR and AraR from Aspergillus species and bacterial repressors XylR and AraR.

Heterologous biosynthesis of betanin triggers metabolic reprogramming in tobacco.

Engineering of a specialized metabolic pathway in plants is a promising approach to produce high-value bioactive compounds to address the challenges of climate change and population growth. Understanding the interaction between the heterologous pathway and the native metabolic network of the host plant is crucial for optimizing the engineered system and maximizing the yield of the target compound. In this study, we performed transcriptomic, metabolomic and metagenomic analysis of tobacco (Nicotiana tabacum) plants engineered to produce betanin, an alkaloid pigment that is found in Caryophyllaceae plants.

Model-based optimization of cell-free enzyme cascades exemplified for the production of GDP-fucose.

Cell-free production systems are increasingly used for the synthesis of industrially relevant chemicals and biopharmaceuticals. Cell-free systems often utilize cell lysates, but biocatalytic cascades based on recombinant enzymes have emerged as a promising alternative strategy. However, implementing efficient enzyme cascades is a non-trivial task and mathematical modeling and optimization has become a key tool to improve their performance.

Adaptive laboratory evolution and metabolic engineering of Cupriavidus necator for improved catabolism of volatile fatty acids.

Bioconversion of high-volume waste streams into value-added products will be an integral component of the growing bioeconomy. Volatile fatty acids (VFAs) (e.g.

Engineering Halomonas bluephagenesis for synthesis of polyhydroxybutyrate (PHB) in the presence of high nitrogen containing media.

The trade-offs exist between microbial growth and bioproduct synthesis including intracellular polyester polyhydroxybutyrate (PHB). Under nitrogen limitation, more carbon flux is directed to PHB synthesis while growth is inhibited with diminishing overall carbon utilization, similar to the suboptimal carbon utilization during glycolysis-derived pyruvate decarboxylation. This study reconfigured the central carbon network of Halomonas bluephagenesis to improve PHB yield theoretically and practically.

Applying metabolic control strategies to engineered T cell cancer therapies.

Chimeric antigen receptor (CAR) T cells are an engineered immunotherapy that express synthetic receptors to recognize and kill cancer cells. Despite their success in treating hematologic cancers, CAR T cells have limited efficacy against solid tumors, in part due to the altered immunometabolic profile within the tumor environment, which hinders T cell proliferation, infiltration, and anti-tumor activity. For instance, CAR T cells must compete for essential nutrients within tumors, while resisting the impacts of immunosuppressive metabolic byproducts.

Generation of a Vibrio-based platform for efficient conversion of raffinose through Adaptive Laboratory Evolution on a solid medium.

Raffinose, a trisaccharide abundantly found in soybeans, is a potential alternative carbon source for biorefineries. Nevertheless, residual intermediate di- or monosaccharides and low catabolic efficiency limit raffinose use through conventional microbial hosts. This study presents a Vibrio-based platform to convert raffinose efficiently.

Engineering peroxisomal surface display for enhanced biosynthesis in the emerging yeast Kluyveromyces marxianus.

The non-conventional yeast Kluyveromyces marxianus is a promising microbial host for industrial biomanufacturing. With the recent development of Cas9-based genome editing systems and other novel synthetic biology tools for K. marxianus, engineering of this yeast has become far more accessible.

A multiscale hybrid modelling methodology for cell cultures enabled by enzyme-constrained dynamic metabolic flux analysis under uncertainty.

Mammalian cell cultures make a significant contribution to the pharmaceutical industry. They produce many of the biopharmaceuticals obtaining FDA-approval each year. Motivated by quality-by-design principles, various modelling methodologies are frequently trialled to gain insight into these bioprocesses.